Nanoparticles up-regulate tumor necrosis factor-α and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation

Toxicology and Applied Pharmacology 238 (2009) 160–169

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Nanoparticles up-regulate tumor necrosis factor-α and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation Hye-Mi Lee a,b, Dong-Min Shin a,b, Hwan-Moon Song c, Jae-Min Yuk a,b, Zee-Won Lee d, Sang-Hee Lee e, Song Mei Hwang f, Jin-Man Kim f, Chang-Soo Lee c,⁎, Eun-Kyeong Jo a,b,g,⁎ a

Department of Microbiology, College of Medicine, Daejeon 301-747, South Korea Infection Signaling Network Research Center, College of Medicine, Daejeon 301-747, South Korea Department of Chemical Engineering, College of Engineering, Chungnam National University, Daejeon 305-764, South Korea d Glycomics Team, Korea Basic Science Institute, Daejeon 305-333, South Korea e Molecular Genomics Laboratory, Department of Biological Science, KAIST, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, South Korea f Department of Pathology, College of Medicine, Daejeon 301-747, South Korea g Research Institute for Medical Sciences, College of Medicine, Daejeon 301-747, South Korea b c

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Article history: Received 28 February 2009 Revised 8 May 2009 Accepted 10 May 2009 Available online 18 May 2009 Keywords: Nanoparticles Inflammation Tumor necrosis factor-α CXCL-8 Reactive oxygen species Mitogen-activated protein kinases

a b s t r a c t Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-α and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-α and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-α and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo. © 2009 Elsevier Inc. All rights reserved.

Introduction Colloidal semiconductor nanoparticles, or quantum dots (QDs), are single crystals with diameters of a few nanometers. Nanoparticle size can be precisely controlled by varying the reaction time, temperature, and ligand molecules. Recently, QDs have emerged as promising fluorescent markers for in vitro and in vivo imaging, because in contrast to organic fluorescence dyes, they have unique optical properties such as bright and photostable fluorophores with a broad excitation but narrow emission wavelength range (Bruchez, 2005; Medintz et al., 2005). The use of QDs for tracking noninvasive intracellular events facilitates basic research on underlying mechanisms as well as improves the clinical translation of basic research ⁎ Corresponding authors. Dr. Eun-Kyeong Jo is to be contacted at Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, South Korea. Dr. Chang-Soo Lee, Department of Chemical Engineering, College of Engineering, Chungnam National University, Daejeon, South Korea. Fax: +82 42 585 3686. E-mail addresses: [email protected] (C.-S. Lee), [email protected] (E.-K. Jo). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.05.010

findings (Kiessling, 2008). However, an important issue concerning the toxicity of nanoparticles has recently arisen, because there is little information about the toxicological effects or detailed biological mechanisms such as the induction of inflammatory toxic responses. Therefore, a study about the toxicity of nanoparticles is essential to validate their biological application in vivo. In general, QDs are composed of periodic groups II–VI (e.g., CdSe) or III–V (e.g., InP) materials (Alivisatos et al., 2005). Although the sizes of QDs can be significantly controlled by synthetic conditions or surface modifications, their diameters can range from 1 to 25 nm (Clift et al., 2008). Recent technical progress in the field of QD development has shown that water-soluble and highly luminescent nanoparticles can be easily obtained (Medintz et al., 2005; Yu et al., 2006; Biju et al., 2008). Nevertheless, much work remains to be done to examine potential inflammatory and cytotoxic properties of QDs in human cell systems and the effects of the size and raw materials of QDs on their potential toxicity. The relatively recent introduction and rapid expansion of the nanotechnology field may contribute to the incidence of adverse

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health effects, although controlled epidemiological studies are basically non-existent (Li et al., 2008). As foreign particles, nanoparticles are able to affect host immune responses such as the release of inflammatory cytokines and matrix metalloproteinases (MMPs) and the generation of reactive oxygen species (ROS) (Wan et al., 2008). A recent study has demonstrated that inflammatory cytokine tumor necrosis factor (TNF)-α mRNA expression was increased significantly in nano-sized carbon black-treated mice (Tin-Tin-Win-Shwe et al., 2008). In addition, titanium dioxide nanoparticles have been shown to induce pulmonary toxicity and to up-regulate chemokines that may be responsible for pulmonary emphysema development and alveolar epithelial cell apoptosis (Chen et al., 2006). The cytotoxicity of QDs can be attributed to the leaching of harmful metals from their nanocrystal core (QD core degradation), their ability to induce generation of ROS, or interactions of QDs with intracellular components leading to loss of function (Hardman, 2006). Redoxresponsive signaling pathways induced by nanoparticle-mediated oxidative stress have been implicated in the development of adverse effects through the activation of ubiquitously expressed mitogenactivated protein kinases (MAPKs) (Beck-Speier et al., 2005; Donaldson et al., 2003). MAPKs are involved in signal transduction via the activation of numerous cellular proteins and transcription factors (Seger and Krebs, 1995). Therefore, the oxidative potential of nanoparticles is an important parameter for evaluating toxicity and triggers of inflammatory or immunological responses in a variety of cells and tissues. Many intracellular targeting studies have revealed that Tat peptide-conjugated QDs are tethered to inner vesicular surfaces and are actively transported along microtubule tracks to microtubule organizing centers (Ruan et al., 2007). A recent study has demonstrated that QDs are endocytosed by dendritic cells and compartmentalized inside the cytoplasm (Sen et al., 2008). In addition, QDs were found to be localized within intracellular vacuoles in human epidermal keratinocytes (Ryman-Rasmussen et al., 2007). Although accumulating data provide new insights into the intracellular uptake and active transport of nanoparticles, it is not clear whether watersoluble QDs would undergo the same processes of cellular uptake and transport. Furthermore, there is very limited knowledge about the potential toxicity of water-soluble QDs (Biju et al., 2008). This study investigated the potential toxicity of water-soluble nanoparticles (mercaptoacetic acid-conjugated CdSe), because QDs are most frequently used in aqueous solution (Aldana et al., 2001). We determined the effects of QDs on the generation of ROS and the production of TNF-α and CXC-chemokine ligand (CXCL) 8 in human primary monocytes. We also investigated the molecular mechanisms of the QD-mediated TNF-α and CXCL8 production by human primary monocytes. QDs clearly induced the phosphorylation of MAPKs, a crucial step for inflammatory responses and cellular activities, in a ROS-dependent manner. The study shows that the internalized QDs are trapped in endocytic vesicles in the cytoplasm before they are trafficked to lysosomal compartments. Finally, in vivo administration of QDs by intravenous injection resulted in an increased recruitment of neutrophils to lung tissues of mice, further suggesting a role for QDs in the induction of inflammatory responses. Materials and methods Synthesis of quantum dots and its surface modification. The QDs (CdSe/ZnS, core-shell nanoparticles) prepared in this work were synthesized in a three-component coordinating solvent mixture of hexadecylamine, trioctylphosphine (TOP), and trioctylphosphine oxide (TOPO) (Bruchez et al., 1998). The ZnS precursor, diethylzinc (CH2CH3)2, was added to hexamethyl (disilanthaine)((TMS)2S) at a 1:1 ratio in TOP, before adding to a TOPO-functionalized CdSe solution at 270 °C. This solution was sequentially washed with methanol and chloroform. The synthetic nanoparticles were stored in chloroform at

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4 °C. The critical step to control particle size was manipulated with reaction times ranging from 30 s to 2 h. In this study, the particles (QD645) were obtained with a reaction time of 90 min. The resulting organic-soluble CdSe/ZnS could be converted into water-soluble nanoparticles through ligand exchange with mercaptoacetic acid (MAA). In brief, the TOPO-capped CdSe/ZnS QDs (5 mg/ ml) were mixed with a 1 M MAA. The transparent mixture was sonicated for 200 min at 60 °C. To eliminate the excess MAA, the aqueous solution of QDs was centrifuged after the addition of phosphate-buffered saline (PBS) (20 mM, pH 7.4). Finally, the recovered water-soluble QDs, with an average size of 10.5 nm, were stored in PBS at 4 °C. Preparations of QDs used in the experiments were tested for lipopolysaccharide (LPS) contamination by a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and contained less than 20 pg/ml at the concentrations of QDs used in the following experiments. Isolation and culture of human monocytes and cell lines. This study was reviewed and approved by the Institutional Research Board of Chungnam National University Hospital, and written informed consent was obtained from each participant. Venous blood was drawn from the healthy subjects into sterile blood collection tubes, and peripheral blood mononuclear cells were isolated by density sedimentation over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated for 1 h at 37 °C, and nonadherent cells were removed by pipetting off the supernatant. Adherent monocytes were collected as previously described (Jung et al., 2006). Human monocyte and THP-1 (ATCC TIB-202) cells were maintained in RPMI 1640 complete medium (Gibco-BRL, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco-BRL), sodium pyruvate, nonessential amino acids, penicillin G (100 IU/ml), and streptomycin (100 μg/ml). THP-1 cells were treated with 4 nM phorbol-12-myristate-13-acetate (Sigma) for 24 h to induce differentiation into macrophage-like cells and then washed three times with PBS. Cell viability assays. Cell viability of human monocytes was determined using a cell count assay kit (CCK8; Dojindo Molecular Technologies, Gaithersburg, MD), which measures the reduction of WST-8, a water-soluble tetrazolium salt, by dehydrogenases in viable cells. Human monocytes (100 μl of 200,000 cells ml− 1) were seeded in each well of a 96-well culture plate and allowed to grow overnight at 37 °C with 5% CO2. AC toxin was added, and the cells were incubated at 37 °C for the indicated times. CCK8 solution (10 μl) was then added and incubated for 1 h at 37 °C. Absorbance was measured at 450 nm using a μQuant microplate reader (Bio-Tek Instruments, Winooski, VT). Viable cells were determined as a percentage of control cells. Antibodies and reagents. Specific antibodies against phospho(Thr202/Tyr204)-extracellular signal-regulated kinases (ERK) 1/2 and phospho-(Thr180/Tyr182)-p38 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-(Ser345)p47phox antibody, as described previously (Yang et al., 2007), was kindly provided by Dr. J. El-Benna (Inserm, Paris, France). LPS (Escherichia coli 026:B6) was purchased from Sigma. Dimethyl sulfoxide (DMSO; Sigma) was added to cultures at 0.1% (v/v) as a solvent control. The specific nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodium (DPI), the ROS scavenger N-acetyl-L-cysteine (NAC), the xanthine oxidase inhibitor allopurinol, the p38 inhibitor SB203580, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125 were purchased from Calbiochem (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. A sandwich ELISA was used to detect human and mouse TNF-α and CXCL8 (BD PharMingen Inc, San Diego, CA, USA) in culture supernatants. Assays were performed as recommended by the

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manufacturer. Cytokine concentrations in the samples were calculated using standard curves generated from recombinant cytokines; the results are expressed in picograms per milliliter. For Western blot analysis, total cell lysates were prepared after treatment with QDs during the time indicated (0–480 min). Antibodies against phosphoERK1/2, phospho-p38, and α-actin were used at 1:1000 dilutions. Immunoreactive proteins were visualized using a chemiluminescence assay (ECL; Pharmacia-Amersham, Freiburg, Germany) and subsequently exposed to X-ray film (Fuji Film, Tokyo, Japan). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Total RNA for semi-quantitative RT-PCR analysis was extracted from cells using TRIzol reagent (Invitrogen Life technology, Carlsbad, CA, USA), as previously described (Yang et al., 2006). The sequences of the primers used were as follows: hTNF-α forward, 5′-CAGAGGGAAGAGTTCCCCAG-3′, and reverse, 5′-CCTTGGTCTGGTAGGAGACG-3′; hCXCL8 forward, 5′-CATGACTTCCAAGCTGGCCG-3′, and reverse, 5′-TTTATGAATTCTCAGCCCTC-3′; and β-actin forward, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′, and reverse, 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′. The PCR amplification program consisted of 35 cycles of denaturation for 30 s at 94 °C and annealing for 30 s at 55 °C for CXCL8, or 66 °C for TNF-α and β-actin, and 30 s extension at 72 °C. For quantitative real-time PCR analysis, cDNA was amplified using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and primers specific for CXCL8 and β-actin, in a real-time PCR machine (Rotor-Gene 2000, Corbett Research, Sydney, Australia). The cycle threshold values, which denote the starting cycle for amplification, were obtained using Rotor-Gene v6.0.

fixed in 10% formalin and sectioned in paraffin. The paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E). Also directly after euthanasia, the major organs, including the lungs, were excised, rinsed in PBS, and embedded in paraffin. As previously described (Tarabishy et al., 2008), 5-μm paraffin sections were deparaffinized and hydrated by serially dipping into 100, 95, and 80% ethanol; distilled water; and PBS. The slides were blocked with 1.5% normal rabbit serum in PBS for 20 min and immunohistochemically stained for neutrophils, using appropriate antibodies (NIMP-R14, Abcam, Cambridge, MA, USA). In vitro imaging of QDs in THP-1 cells. To observe the uptake of QDs, THP-1 cells were seeded on coverglass chambers and incubated with medium containing 20 nM QD. Unless otherwise noted, the temperature was maintained at 37 °C. Using a LSM510 confocal microscope (Carl Zeiss, Jena, Germany), QD fluorescence within regions containing a single cell was measured at various time points (0–120 min). Fluorescence intensity is presented as the ratio of QD fluorescence to that of untreated control cells. For lysosomal colocalization, harvested THP-1 cells were cultured overnight on coverglass chambers and then incubated with medium containing 20 nM QDs for various times at 37 °C. Subsequently, THP-1 cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and then stained with a lysosome tracker (Lyso-tracker™, Molecular Probes), according to the manufacturer's instructions. The cells were washed with PBS, dehydrated in 100% ethanol, and mounted using Fluoromount-G (Southern Biotech, Birmingham, AL, USA). The fixed cells were imaged using an LSM510 confocal microscope (Zeiss).

Measurement of intracellular ROS. Intracellular ROS levels were detected by a dihydrorhodamine-1,2,3 (DHR) assay, in which ROS convert dihydrorhodamine to rhodamine, and measured spectrofluorometry, as described previously (Rothe et al., 1991). In brief, human primary monocytes (1 × 106 ml) were incubated in Hanks' balanced salt solution (132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM potassium phosphate, 20 mM HEPES, 5.5 mM glucose, and 0.5% w/v BSA, pH 7.4) plus 1 mm CaCl2 for 5 min at 37 °C in a shaking water bath, and then DHR (5 μM; Molecular Probes, Eugene, OR) was added. Catalase (Sigma; 0.2 mg/ml) was added to avoid carry-over of hydrogen peroxide from NADPH oxidase-active cells to inactive cells. After DHR loading, the cells were stimulated with QDs (5 nM) for various times (0–240 min). The reaction was stopped with an addition of 2 ml of icecold PBS containing 1% (v/v) BSA. Samples were kept on ice until analysis in a spectrofluorometer (Molecular Devices, Sunnyvale, CA). The peak excitation and emission wavelengths were 500 and 536 nm, respectively. The QDs did not interfere with the spectra.

Electron microscopy. Harvested THP-1 cells were incubated with medium containing 20 nM QDs for various times and processed as described previously (Zhang et al., 2008). Briefly, THP-1 cells were fixed with 2% glutaraldehyde (Electron Microscopy Sciences, Road Hatfield, PA, USA) in 0.1 M sodium cacodylate, pH 7.3 (Ted Pella™, Redding, CA, USA) for 20 min, washed with 0.1% cacodylate buffer, and postfixed with 1% osmium tetroxide solution (Electron Microscopy Sciences) for 30 min. Subsequently, the THP-1 cells were stained with 4% uranyl acetate for 10 min. A Tecnai G2 Spirit Twin transmission electron microscope (FEI Company, USA) and a JEM ARM 1300S highvoltage electron microscope (JEOL, Japan) were used.

Determination of NADPH oxidase activity. NADPH oxidase activity was measured using a lucigenin (bis-N-methylacridinium nitrate) chemiluminescence assay (5 × 10− 6 mol/L lucigenin, Sigma) in the presence of its substrate NADPH (10− 4 mol/L, Sigma), as described previously (Griendling et al., 1994). Chemiluminescence occurred within 1 min after the addition of 100 μM NADPH and was recorded using a luminometer (Lumet LB9507; Berthold Technologies, Bad Wildbad, Germany). The emitted light intensity was normalized to a blank and used as a measure of superoxide production.

Results

Histology and immunohistochemistry. All animals were maintained in a pathogen-free environment. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chungnam National University. BALB/c mice were given 2 nmol QDs/mouse in a total volume of 50 μl of sterile saline once in a day for 0, 3, 5 days via intravenous (i.v.) injection. In the negative control animals, the same volume of saline solution was injected. In the positive control animals, LPS (20 mg/kg) was injected. After 24 h, the animals were euthanized to obtain lung tissue. The samples were

Statistical analysis. The data obtained from independent experiments are presented as the mean ± standard deviation (SD). Comparisons were analyzed using a paired t-test with a Bonferroni adjustment, or ANOVA for multiple comparisons. Differences were considered significant at P b 0.05.

In vitro cytotoxic effects of QDs for human primary monocytes The cytotoxic effects of QDs on human primary monocytes have not been investigated, although cell viability and toxicity in the presence of QDs have been studied for various cell types (Chan and Kan, 1999; Zhang et al., 2008; Ryman-Rasmussen et al., 2007). First, the dose-dependent cytotoxic effects of QDs on human primary monocytes were evaluated in the present study. In vitro cytotoxicity was evaluated with CCK8 assays after culturing the primary monocytes in the presence of QDs for 24 h. As shown in Fig. 1, the QDs had no significant effects on cell viability below QD concentrations of 10 nM. Cell viability significantly decreased by 88% (P b 0.001) in human primary monocytes following treatment with 25 nM QDs. The cytotoxicity of QDs was enhanced with increases in nanoparticle concentration (Fig. 1). Cell viability decreased by 70% at a QD concentration of 100 nM (Fig. 1). These results indicate that QDs induce toxic effects on cellular viability of human primary monocytes

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Fig. 1. Effect of QDs on cell survival in human primary monocytes, based on CCK8 assays. Cells were treated with different concentrations (0.04–100 nM) of QDs for 24 h. Data are expressed as percentage reduction of CCK8 and were calculated by dividing the optical density (OD) of supernatants for each treatment well by the average OD from all corresponding supernatants from control wells (×100). Cell viability for controls was N 95%. The quantitative data for cell viability is presented as the mean ± SD of three experiments. ⁎⁎⁎ P b 0.001 compared with control cultures.

at concentrations greater than 25 nM. Therefore, we used a concentration of 5 nM of QDs in the following experiments.

QDs induced mRNA and protein expression of TNF-α and CXCL8 in human primary monocytes Previous studies have demonstrated that carboxylic acid-coated QDs significantly increased the production of interleukin (IL)-1β, IL-6, and CXCL8 in epidermal keratinocytes (Ryman-Rasmussen et al.,

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2007). However, there is little information of the immunotoxicity of QDs in monocytes/macrophages. To examine whether QDs activate the pro-inflammatory cytokine TNF-α or affect chemokine production in human primary monocytes, the cells were treated with the indicated doses of QDs and cultured for various times. QD stimulation of primary monocytes induced the up-regulation of TNF-α and CXCL8 mRNA expression in a dose-dependent manner, as measured by semiquantitative RT-PCR analysis (Fig. 2A). In addition, the kinetics showed that TNF-α and CXCL8 mRNA expression was induced by QDs beginning at 3 h and peaking at 6 h post-treatment (Figs. 2B and C, semi-quantitative and real-time RT-PCR analysis, respectively). Treatment of monocytes with QDs resulted in a robust activation of TNF-α and CXCL8 secretion, both of which peaked at 18 h (Fig. 2D). Notably, the QD-induced levels of mRNA and protein expression of TNF-α and CXCL8 were comparable to those induced by LPS (Figs. 2B– D, mRNA and protein, respectively). These findings suggest that QDs actively induce TNF-α and CXCL8 production in human primary monocytes. QDs actively induce ROS generation in human primary monocytes A previous study reported that CdSe nanoparticles induced apoptosis and increased ROS generation in human neuroblastoma cells (Chan et al., 2006). To investigate whether QDs can induce ROS generation in human primary monocytes, we treated the cells with QDs for various times (0–240 min) and analyzed ROS generation with DHR assays (Fig. 3A). Quantification data show that ROS production was significantly increased in human monocytes after QD treatment

Fig. 2. Expression of TNF-α and CXCL8 mRNA and protein in human primary monocytes treated with QDs. Human primary monocytes were treated with QDs at different concentrations (0.2–25 nM, Panel A) or for different times (3–72 h, Panels B to D). Cells were harvested at 6 h (Panel A) and subjected to semi-quantitative RT-PCR (Panels A and B), real-time RT-PCR (Panel C), or ELISA analysis (Panel D) for TNF-α and CXCL8. (A and B) The expression level of β-actin mRNA was used as a control. Representative gel images of three independent experiments with similar results are shown. (C and D) The quantitative data for mRNA (Panel C) and protein (Panel D) expression of TNF-α and CXCL8 are shown as the mean ± SD of three experiments. U, untreated; L, LPS.

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nearly abolished by pre-treatment with DPI. These results demonstrate that QDs actively induce NADPH oxidase-dependent ROS generation in human primary monocytes. NADPH oxidase-dependent ROS release is involved in QD-induced production of TNF-α and CXCL8 in human primary monocytes Recent studies have shown that CXCL8 and macrophage inflammatory protein-1β expression in human monocytic cell line U937 is dependent on a ROS-dependent pathway (Higai et al., 2007; Higai et al., 2008). To confirm that ROS generation is involved in QD-induced expression of TNF-α and CXCL8 mRNA and protein in human primary monocytes, the cells were pre-treated with or without ROS scavengers. Then, the mRNA and supernatants were collected to assess mRNA and protein expression of TNF-α and CXCL8. Pre-treatment with a general ROS scavenger (NAC) or a NADPH oxidase inhibitor (DPI) dose-dependently attenuated QD-induced TNF-α and CXCL8 mRNA and protein expression in human monocytes (Figs. 4A–C; RTPCR analysis, Panels A and B; ELISA analysis, Panel C). In contrast, pretreatment with allopurinol, a xanthine oxidase inhibitor, did not affect the mRNA or protein expression of TNF-α and CXCL8 in human primary monocytes (Figs. 4A–C). These data indicate that QD-induced TNF-α and CXCL8 expression in human primary monocytes are at least partly mediated via ROS generated by NADPH oxidase. QD-dependent MAPK activation is involved in the expression of TNF-α and CXCL8 in human primary monocytes

Fig. 3. QD-mediated ROS generation in human primary monocytes. Human primary monocytes were treated with QDs (5 nM) or LPS (100 ng/ml) for the indicated times (0–240 min, Panels A and B). (A) The cells were then washed and incubated with 5 μM DHR123 for 30 min. Fluorescence was measured using a spectrofluorometer. The quantitative data for the relative DHR fluorescence intensities are shown as the mean ± SD of three experiments. (B) The cells were harvested and subjected to Western blot analysis to detect phosphorylated p47phox at Ser345. The same blots were washed and re-blotted for α-actin as a loading control. Data are representative of three independent experiments. Densitometric analysis of data for three independent experiments (means ± SD) is shown (Bottom). The densitometry values for phosphorylated p47phox were normalized to the α-actin level. (C) NADPH oxidase activity was measured by a lucigenin-derived chemiluminescence assay. The effect of the NADPH oxidase inhibitor DPI (20 μM) was also examined. Data are the mean ± SD of all cases (n = 5). ⁎⁎⁎P b 0.001 compared with control cultures treated with solvent.

(Fig. 3A). The kinetics of QD-induced ROS generation was similar to those of LPS-induced ROS generation, both reaching a peak by 30 min (Fig. 3A). The phosphorylation of p47phox is a key step in NADPH oxidase activation, and p47phox phosphorylation at Ser345 is essential to induce the priming of ROS production (Dang et al., 2006). To assess the involvement of NADPH oxidase in the increased cellular capacity to generate ROS, we examined the Ser345 phosphorylation of p47phox, the cytosolic component of NADPH oxidase, at different time points. Fig. 3B shows that p47phox phosphorylation at Ser345 actively increased in human primary monocytes and reached a maximal level at 5 to 15 min after stimulation with QDs. When NADPH oxidase activity was measured in human primary monocytes via lucigenin chemiluminescence, the QD-treated cells showed robustly increased NADPH oxidase activity, similar to the levels induced by LPS stimulation (Fig. 3C). The NADPH oxidase activity was

MAPK activation plays an essential role in macrophage responses to pro-inflammatory stimuli such as cytokines (Xiao et al., 2003; Chan and Kan, 1999; Li et al., 2004; Donaldson et al., 2003). We examined the possibility of MAPK activation by QDs in human primary monocytes by treating the cells with 5 nM of QDs for various times and subjecting them to Western blot analysis. As shown in Fig. 5A, the QDs actively induced the phosphorylation of MAPKs (ERK1/2 and p38 MAPK) within 5 min of stimulation, and the peak activation was observed at around 30 to 60 min. The QDs did not induce the phosphorylation of JNK in human monocytes (data not shown). To confirm that ROS generation affected the QD-induced MAPK activation, human primary monocytes were pre-treated with or without ROS scavengers. Then, the MAPK activation was assessed by Western blotting. Pre-treatment with a general ROS scavenger (NAC) or a NADPH oxidase inhibitor (DPI), but not the xanthine oxidase inhibitor allopurinol, caused dose-dependent attenuation of the QD-induced MAPK activation in human monocytes (Fig. 5B). These data suggest that QD-induced MAPK activation is dependent on ROS generation in monocytes. To assess the role of MAPKs related to TNF-α and CXCL8 expression, cells were pre-treated with various MAPK inhibitors, including the p38 inhibitor SB203580, the MEK inhibitor U0126, and the JNK inhibitor SP600125, for 45 min prior to the addition of QDs (5 nM) and then subjected to RT-PCR (semi-quantitative and realtime RT-PCR, Panels C and D, respectively) and ELISA (Panel E). Pretreatment with the p38 and MEK inhibitors, but not the JNK inhibitor, significantly inhibited QD-mediated expression of TNF-α and CXCL8 in human primary monocytes in a dose-dependent manner (Figs. 5C– E). These data indicate that both the p38 MAPK and ERK1/2 pathways are critical in QD-mediated TNF-α and CXCL8 expression in human monocytes. Intracellular uptake and trafficking of QDs by human macrophages Recently, dendritic cells were shown to endocytose streptavidinconjugated or unconjugated QDs, which were compartmentalized in cytoplasmic vesicles and were trafficked later to lysosomes (Sen et al., 2008). We further examined the intracellular localization of QDs in

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Fig. 4. ROS involvement in QD-induced TNF-α and CXCL8 expression. (A–C) Human primary monocytes were pre-treated with or without NAC (10, 20, or 30 mM), DPI (5, 10, or 20 μM), or allopurinol (0.01, 0.1, or 1 mM) for 45 min, and then treated with QDs (5 nM). (A and B) Cells were harvested at 6 h after treatment with QDs. Total RNA was collected and subjected to semi-quantitative (A) and real-time RT-PCR (B) analysis for TNF-α and CXCL8 mRNA expression. The mRNA expression level of β-actin was used as a control. A representative gel of three independent replicates with similar results is shown (Panel A). The quantitative data for mRNA (B; at 6 h) and protein (C; at 18 h) expression of TNF-α and CXCL8 are shown as the mean ± SD of three experiments. ⁎⁎⁎P b 0.001 compared with solvent control cultures. U, untreated; D, solvent control (0.1% DMSO).

human monocytic THP-1 cells. To detect internalization of QDs, we investigated the kinetics of QD uptake following the addition of QDs to the medium. Confocal microscopy revealed that QD uptake by THP-1 cells occurred at various times after incubation with QD-containing medium at 37 °C. Within 1 min, QD fluorescence appeared as a band adjacent to the nearby plasma membrane in THP-1 cells (data not shown). Afterward, distinct QD-containing vesicles were apparent inside the cells (Fig. 6, 15 min). Previous studies have found QDs colocalized with lysosomes after 45 min (Sen et al., 2008). We also determined whether QD-containing vesicles colocalized with lysosomes by using a specific lysosomal tracker. In THP-1 cells, QDcontaining vesicles did not colocalize with lysosomes within 15 to 60 min of incubation (Fig. 6A). However, at later time points (N60 min), an increasing number of QD-containing vesicles colocalized with lysosomes (Fig. 6A), indicating that QDs are retained inside lysosomes. We also visualized the distribution of QDs inside THP-1 cells by electron microscopy. As shown in Fig. 6B, endocytosed QDs were present in the cytoplasm and were compartmentalized inside lysosomes. We also examined whether lysosomal accumulation of QDs triggered the inflammatory reaction. To answer this question, we pre-treated primary monocytes with bafilomycin A or chloroquine to prevent endosomal acidification prior to the QD treatment. The bafilomycin A and chloroquine treatments significantly decreased the secretion of TNF-α and CXCL8 induced by QDs in monocytes (Fig. 6C).

Collectively, these data indicate that QDs are endocytosed via cytoplasmic vesicles and are eventually colocalized with lysosomes in human monocytes. In addition, endosomal acidification after the internalization of QDs is required for the inflammatory cytokine and chemokine production that occurs in response to QDs. In vivo infiltration of neutrophils in the lungs and increased levels of CXCL8 and TNF-α in sera from QD-injected mice Neutrophil migration to inflammatory sites can be induced by various inflammatory mediators such as CXCL8, macrophage inflammatory protein-2 (CXCL2), and KC (CXCL1) (Frangogiannis, 2004). These cytokines provide the first and most potent cellular line of innate host defense (Dallegri and Ottonello, 1997). TNF-α expression also precedes the infiltration of inflammatory cells into the injured zone (Feuerstein et al., 1994). To examine the in vivo inflammatory reactions to QDs, mice were divided into three groups: saline-injected (negative control), QD-injected (2 nmol/mouse), and LPS-injected (20 mg/kg; positive control) groups. QDs were intravenously injected into the mice at three different conditions (once a day; 1, 2, or 3 injections). Four major organs (liver, lung, spleen, and kidneys) were removed from each mouse, and microscopic examinations were performed after a general H&E staining process. The livers, kidneys, and spleens from QD-injected mice revealed rare focal pathological findings similar to those in the control group. The only pathological

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Fig. 5. QD-dependent MAPK activation is required for TNF-α and CXCL8 expression in human primary monocytes. (A and B) Western analysis for QD-mediated MAPK activation. (A) Human primary monocytes were treated with QDs (5 nM) for the indicated times. (B) Human primary monocytes were pre-treated with or without NAC (10, 20, or 30 mM), DPI (5, 10, or 20 μM), or allopurinol (0.01, 0.1, or 1 mM) for 45 min, and then treated with QDs (5 nM) for 30 min. The cells were harvested and subjected to Western blot analysis for phosphorylated p-p38 and p-ERK1/2. The same membranes were stripped and reprobed for α-actin as a loading control. The data are representative of three independent experiments with similar results. (C–E) Human primary monocytes were pre-treated with or without SB203580 (1, 5, or 10 μM), U0126 (5, 10, or 20 μM), or SP600125 (5, 20, or 30 μM) for 45 min and then treated with QDs (5 nM). The mRNA and supernatants were collected at 6 h and 18 h, respectively. Semi-quantitative (C) and real-time RT-PCR (D) analyses were performed to determine TNF-α and CXCL8 mRNA expression. The mRNA expression level of β-actin was used as a control. A representative gel of three independent replicates with similar results is shown (Panel C). The quantitative data for mRNA (D; at 6 h) and protein (E; at 18 h) expression of TNF-α and CXCL8 are shown as the mean ± SD of three experiments. ⁎⁎⁎P b 0.001 compared with solvent control. U, untreated; D, solvent control (0.1% DMSO).

finding observed by H&E staining was an increased neutrophil infiltration in the lungs of the control mice and the QD-injected mice (data not shown). Therefore, we performed immunohistochemical analysis using anti-mouse neutrophil antibodies. As shown in Fig. 7A, the histopathological data show that the repeated intravenous injection of QDs (2 nmol/mouse/3 days) markedly induced neutrophil infiltration into the lung, which was comparable to the infiltration induced by intraperitoneal injection of LPS (20 mg/kg). We next evaluated the levels of CXCL8 and TNF-α in lung tissues and sera from QD-injected mice. When the mice were injected with QDs, CXCL8 and TNF-α were significantly up-regulated to levels matching those of the mice injected with LPS (Fig. 7B, for lung tissues;

Figs. 7C and D, for sera). The repetitive injection of QDs significantly increased the levels of CXCL8 and TNF-α, compared with the levels in saline-injected control mice (Figs. 7C and D). Taken together, these data confirm that intravenously injected QDs specifically induce neutrophil migration into the lungs and up-regulate TNF-α and CXCL8 levels in the lung tissues and sera of mice. Discussion QDs offer the invaluable power of high-end multicolor technology and improved detection sensitivity for immune markers in multicolor flow cytometry (Chattopadhyay et al., 2006). In particular, CdSe/ZnS

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Fig. 6. QD uptake and intracellular localization in human monocytic cells. (A) Confocal images showing colocalization of QD-containing vesicles with lysosomes, using a lysosomal tracker, at different times of incubation with QDs. Images show lysosomes (green), QDs (red), and overlays. Scale bar = 10 μm. (B) Ultrastructure of QD uptake images. THP-1 cells were incubated with QDs for 2 h. Indicated regions (a and c) are shown at higher magnification in b and d, respectively. Arrows indicate the internalized QDs in lysosomal structures. Scale bars in a and c = 2 μm and in b and d = 1 μm. Lyso, Lysosome tracker; DIC, differential interference contrast images showing the presence of cells on a coverslip. (C) Human primary monocytes were pre-treated with or without bafilomycin A (Baf A; 10, 20, or 50 nM) or chloroquine (Chloro; 1, 5, or 10 μM) for 45 min and then treated with QDs (5 nM). The supernatants were collected at 18 h. The quantitative data for protein expression of TNF-α and CXCL8 are shown as the mean ± SD of three experiments. ⁎⁎P b 0.01 compared with solvent control. U, untreated; D, solvent control (0.1% DMSO).

nanoparticles are one of the most versatile nanomaterials and have a wide range of biological applications (Portney and Ozkan, 2006; Lewinski et al., 2008). In general, QD nanoparticles range from 2 to 100 nm in diameter and typically consist of a core-and-shell structure (Derfus et al., 2004; Lewinski et al., 2008). The core is composed of elements from groups II–VI (e.g., CdSe, CdTe, CdS, PbSe, ZnS, and ZnSe) and groups III–V (e.g., GaAs, GaN, InP, and InAs) (Chan et al., 2002). The cytotoxicity of QD particles is thought to be related to the leaching of core metals, even at low concentrations (Lewinski et al., 2008). Therefore, it is necessary to precisely evaluate the potential harmful effects of nanoparticles on human primary mononuclear cells, as they are the first line of defense in the innate immune system of the body.

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The present study demonstrates that the robust activation of TNFα and CXCL8 expression by human monocytes in response to QDs occurs in a dose- and time-dependent manner. These data partially correlate with recent findings using anionic (carboxylic acid)-coated QDs, which significantly increased the release of IL-1β, IL-6, and CXCL8 by human epidermal keratinocytes (Ryman-Rasmussen et al., 2007). The same study reported a modest but significant cytotoxicity in keratinocytes treated with QD655, suggesting that QDs noxiously affect cells (Ryman-Rasmussen et al., 2007). Recent studies have revealed that QD621-PEG, a nail-shaped QD with a hydrodynamic size of 39–40 nm, can penetrate the skin and induce an inflammatory response, with a significant increase in IL-6 and CXCL8, in human keratinocytes (Zhang et al., 2008). Other studies have shown that metal particles such as nano-Co and nano-TiO2 can alter the transcription and activity of MMP-2 and MMP-9 in human monocytic U937 cells (Wan et al., 2008). In addition, the direct exposure of vascular endothelium to ultrafine particles (b100 nm) elicited an inflammatory response that was dependent on the composition of the particles. In particular, zinc oxide (ZnO) nanoparticles were cytotoxic, leading to considerable cell death (Gojova et al., 2007). Together with our data, these findings indicate that leaching of core materials from QDs may significantly affect primary determinants of inflammatory toxicity in human cells. Previous studies have indicated that ROS and oxidative stress are valid criteria for evaluating nanoparticle toxicity, as both play important roles in the induction of nanoparticle-associated injuries (Li et al., 2008; Nel et al., 2006; Xia et al., 2006). ROS generation by nanomaterials may lead to possible adverse biological effects and oxidative injury to proteins, lipids, and membranes (Nel et al., 2006; Li et al., 2008). In addition, ROS may function as second messengers during inflammatory signaling (Torres and Forman, 2003). Oxidative stress related to carbon nanoparticles has been linked to calciumdependent pathways (Stone et al., 2000) and to pro-inflammatory effects and nuclear factor-κB activation in macrophages (Brown et al., 2004). Previously, nanoparticle-induced MMP release has been shown to be regulated by activator protein 1 and the protein tyrosine kinase signaling pathways associated with redox signaling (Wan et al., 2008). However, the molecular mechanisms of oxidative stress and inflammatory toxicity induced by QDs remain largely unknown. The current data show that QDs elicit a robust activation of intracellular ROS and NADPH oxidase activities. In addition, QDinduced ROS generation was required for the enhanced TNF-α and CXCL8 expression via MAPK activation. These results correlate with previous findings in a cultured bronchial epithelial cell line, BEAS-2B, which showed increased expression of inflammation-related genes, including CXCL8, through p38 MAPK and ERK pathways after exposure to titanium dioxide nanoparticles (Park et al., 2008). QDs generate ROS in the extracellular environment and intracellularly (Lovrić et al., 2005). CaSe QDs can produce singlet oxygen in vitro with exposure to light, and consequently can be used to sensitize a photodynamic therapy sensitizer (Samia et al., 2003). Although we tried to perform our experiments in the absence of light in our system, ROS can form in an aqueous medium (e.g., cell culture medium), since the medium is supplied with exogenous CO2/O2 or a minimum of light via the inverted microscope (Lovrić et al., 2005). The subcellular organelles are the main sensors of oxidative damage (Lovrić et al., 2005). Our data demonstrated that QDs activated NADPH oxidase activity, superoxide generation, and the phosphorylation of p47phox (see Fig. 3). We also initially observed QDs near the plasma membrane (see Fig. 6A). These data strongly suggest that NADPH oxidase plays a role in triggering ROS in response to QD uptake. NADPH oxidase is a membrane-bound enzyme complex found primarily in the plasma and phagosome membranes. The released ROS can cause plasma membrane damage and injure intracellular organelles, including mitochondria, eventually leading to cell death (Lovrić et al., 2005). These data suggest that the QDs near plasma membranes can induce

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Fig. 7. In vivo injection of QDs up-regulates neutrophil infiltration in lung tissues and serum levels of TNF-α and CXCL8 in mice. To examine in vivo inflammatory toxicities, mice were divided into three groups: negative control (saline-injected), QD-injected (2 nmol/mouse), or LPS-injected positive control (20 mg/kg) groups. QDs were intravenously injected into the mice according to three different schedules (once a day; 1, 2, or 3 injections). (A) Immunohistochemistry for neutrophils in the lung tissues. Magnification × 200. Specific quantitation of neutrophil infiltration per each group was analyzed in 6 random fields. The data are presented as the mean ± SD of four experiments. (B) Real-time RT-PCR analyses were performed to determine TNF-α and CXCL8 mRNA expression. Total RNA samples were isolated from lung tissues of the mice groups. The QD-treated mice were given QDs by intravenous injection over 3 days (for Panels A and B). The results are shown as the mean ± SD of four mice. (C and D) Serum levels of CXCL8 (C) and TNF-α (D) 24 h after treatment with saline, QDs, or LPS (n = 4 per group), as measured by ELISA.

ROS generation via NADPH oxidases, resulting in potentially adverse biological responses. The present study indicates that the water-soluble QDs traffic to intracytoplasmic vesicles destined to become lysosomes. Our data agree well with recent studies showing that QD655 is preferentially endocytosed by dendritic cells and compartmentalized inside lysosomes at later times (∼ 45 min) (Sen et al., 2008). Intracellular trafficking studies suggest that QDs affect intracellular physiological functions; QD nanoparticles can be arrested within endosomes and perturb the normal endosomal sorting of the cells (Tekle et al., 2008). The titanium dioxide nanoparticles were taken up into the cytoplasm by the cultured BEAS-2B cells and were localized to the peri-nuclear region as aggregated particles (Park et al., 2008). We found that pretreatment with bafilomycin A or chloroquine inhibited the inflammatory cytokine and chemokine production induced by QDs in monocytes. Previously, pre-treating neutrophils with the endosomal acidification inhibitors bafilomycin A or chloroquine was demonstrated to markedly inhibit CpG-DNA-induced CXCL8 and IL-6 production (József et al., 2006). In addition, the synthesis of TLR3dependent CCL11 was markedly inhibited by bafilomycin A1 (Niimi et al., 2007), supporting the essential role played by the acidification of endosomes in cytokine/chemokine secretion. Together, these data suggest that endosomal acidification after the internalization of QDs is a necessary step for initiating intracellular inflammatory signaling for the production of cytokines and chemokines. There are a growing number of in vitro studies on nanoparticle cytotoxicity using different cell lines, incubation times, and surface coating manipulations (Lewinski et al., 2008). More information about the potential toxicity of nanoparticles in vivo is needed to determine whether the observed cytotoxicity is physiologically relevant (Lewinski et al., 2008). Previous studies on QD deposition and toxicity using animal models have suggested that QDs are relatively nontoxic, because the animals continued to live after QD injection; however, QDs remain fluorescent for at least 4 months in vivo (Ballou et al.,

2004; Lewinski et al., 2008). In the current study, intravenously injected QDs caused increased levels of CXCL8 and TNF-α in the serum of the injected mice, and these levels were similar to those in the serum of LPS-injected mice. In addition, significantly increased neutrophil infiltration was observed in lung tissues of the injected mice, although no significant changes were detected in other major organs, including the liver, spleen, and kidneys, when compared with control mice. Experimental evidence revealed that carbon black nanoparticles intensively aggravated LPS-induced lung inflammation and increased the expression of pro-inflammatory cytokines such as IL-1β and macrophage inflammatory protein-1α (Inoue et al., 2006). The rapid progress of nanotechnology may introduce potential hazards associated with cellular and animal pulmonary toxicity. Exposure to engineered nanoparticles may result in higher risks for pulmonary injury and illness through novel mechanisms, including oxidant injury and pro-inflammatory effects (Li et al., 2008). The current study clearly demonstrates that water-soluble QDs induced robust activation of TNF-α and CXCL8 expression through ROS- and MAPK-dependent pathways in human primary monocytes. Our data also show that internalized QDs are trapped in cytoplasmic vesicles and colocalize in lysosomal compartments. Furthermore, repetitive intravenous injection of QDs resulted in increased recruitment of neutrophils to lung tissues and up-regulated the levels of TNFα and CXCL8 in lung tissues and sera of mice. These data suggest that QDs can induce inflammatory responses in vitro and in vivo. Accumulating evidence will provide a basis for understanding the concurrent toxicity and bioaccumulation of nanoconductor QDs, which can contribute to adverse health effects. Acknowledgments This work was supported by grants from the Korea Science and Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National

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